Process for extraction and purification of lutein, zeaxanthin and rare carotenoids from marigold flowers and plants

ABSTRACT

A process for simultaneously extracting, saponifying, and isolating lutein and zeaxanthin, and a mixture of several rare carotenoids in high purity from plants without the use of harmful organic solvents. Lutein crystals containing 5% zeaxanthin were obtained from the dried petals of Marigold flowers  Tagete erecla  while zeaxanthin was isolated and purified from the berries of  Lycium Chinese Mill  (LCM berries). Similarly, this process has been employed to isolate and purify a mixture of lutein, beta-carotene, neoxanthin, violaxanthin, and lutein epoxide from green plants, preferably, kale, collard green, and spinach. These plants, to a lesser extent, also serve as a source of several rare carotenoids such as alpha-cryptoxanthin (Marigolds) and beta-cryptoxanthin (LCM berries). The purified carotenoids isolated by this process are free from impurities and serve as a safe source of nutritional supplement for human consumption as well as providing a suitable and effective color additive for human foods.

This application is a 371 of PCT/US 98/2229 filed Oct. 21, 1998.

1. FIELD OF THE INVENTION

A simultaneous process for extraction, isolation, and purification oflutein, zeaxanthin, and several rare carotenoids from Marigold flowers,Lycium Chinese Mill, and green plants.

2. BACKGROUND OF THE INVENTION

Carotenoids are amongst the most widespread of the naturally occurringgroups of pigments and are found in all families of the plant and animalkingdoms. To date, as many as seven hundred carotenoids have beenisolated from various sources and their chemical structures have beencharacterized. Numerous epidemiological studies in various populationshave shown that consumption of substantial amounts of fruits andvegetables rich in carotenoids reduces the risk of acquiring severaltypes of cancers. As a result, for nearly two decades, scientists havebeen focussing most of their attention on investigating the protectiveeffect of beta-carotene in prevention of cancer, cardiovascular and eyediseases. This is despite the fact that beta-carotene is only one of theprominent carotenoids found in fruits and vegetables whose consumptionhas been associated with health benefits in humans. The reasons for suchan intense focus can be attributed to the pro-vitamin A activity ofbeta-carotene and the lack of commercial availability of other prominentfood carotenoids.

During the past decade, the author and coworkers have isolated,identified, and quantified carotenoids from fruits and vegetablescommonly consumed in the U.S. These studies have revealed that as manyas 40 to 50 carotenoids may be available from the diet and absorbed,metabolized, or utilized by the human body (Khachik et al. 1991, PureAppl. Chem., 63: 71-80). However, among these, only 13 carotenoids and12 of their stereoisomers are routinely found in human serum and milk(Khachik et al. 1997, Anal. Chem. 69:1873-1881). In addition, there are8 carotenoid metabolites and one stereoisomer in human serum or plasmawhich result from a series of oxidation-reduction reactions of threedietary carotenoids, namely, lutein, zeaxanthin, and lycopene. Thesemetabolites were first isolated and characterized by Khachik et al.(1992, Anal. Chem. 64: 2111-2122). In another study, the ingestion ofpurified supplements of dietary (3R,3′R,6′R)-lutein and(3R,3′R)-zeaxanthin was shown to not only result in an increase in theblood levels of these compounds in humans but also increased theconcentration of their oxidative metabolites in plasma (Khachik et al.1995, J. Cellular Biochem. 22:236-246). These findings, for the firsttime, provided preliminary evidence for the long standing hypothesisthat carotenoids may function as antioxidants in disease prevention. Inaddition, these results also established the importance of non-vitamin Aactive dietary carotenoids, particularly, lutein, zeaxanthin, andlycopene.

In 1985 and 1993, Bone et al. (1985, Vision Res. 25: 1531-1535; 1993,Invest. Ophthalmol. Vis. Sci. 34: 2033-2040) elegantly demonstrated thatthe human macular pigment is a combination of lutein and zeaxanthin andspeculated that these dietary carotenoids may play an important role inthe prevention of an eye disease, namely, Age-Related MacularDegeneration (ARMD). This was later confirmed in a case-controlledepidemiological study in which the high consumption of fruits andvegetables, rich specifically in lutein and zeaxanthin, was correlatedto a 43% lower risk of ARMD (Seddon et al. 1994, J. Am. Med. Assoc. 272:1413-1420). More recently, in addition to lutein and zeaxanthin, theauthor and his co-workers reported on the isolation and identificationof one major and several minor oxidation products of lutein andzeaxanthin in human and monkey retinas (Khachik et al. 1997, J. Invest.Ophthalmol. Vis. Sci. 38:1802-1811). The authors then proposed ametabolic pathways for these compounds which may play an important rolein the prevention of ARMD. Therefore the commercial production of thepurified forms of dietary carotenoids in general, particularly luteinand zeaxanthin, is of great importance. These carotenoids may be used,individually or in combination, as nutritional supplements and foodcolorants as well as in clinical trials where their potential healthbenefits in the prevention of ARMD and cancer can be investigated.

Although lutein and zeaxanthin may be obtained from certain fruits andvegetables, the isolation of lutein from extracts of marigold flowersand zeaxanthin from berries of Lycium Chinese Mill (LCM berries) provesto be most economical. In Marigold flowers lutein is the majorcarotenoid and is normally accompanied by about 3-6% zeaxanthin; in LCMberries zeaxanthin is the major carotenoid and is completely free fromlutein. In both of these plants, lutein and zeaxanthin are esterifiedwith fatty acids such as lauric, myristic, and palmitic acids. Thepurification of lutein fatty acid esters from marigold flowers waspatented by Philip in 1977 (U.S. Pat. No. 4,048,203). However, dietarycarotenol fatty acid esters, in general, have not been detected in humanplasma or serum. Therefore, upon ingestion of purified lutein fatty acidesters by humans, these compounds partially undergo hydrolysis in thepresence of pancreatic secretions in the small intestine to regeneratefree lutein which is then absorbed [Khachik et al. Pure & Appl. Chem.,63(1): 71-80, 1991]. Since the most abundant dietary form of lutein isnot its esterified form, a commercial process that could provide luteinfree from fatty acids was needed.

A method for the purification of free lutein from extracts of marigoldswas first reported in 1991 [Tyzkowski and Hamilton, Poultry Sci., 70(3):651-654, 1991]. However because this method was extremelytime-consuming, used harmful organic solvents, and produced poor yields,it could not be implemented commercially.

In view of the important biological activity of lutein and zeaxanthin,the author developed a process for isolation, purification, andrecrystallization of lutein from saponified Marigold oleoresin which waspatented in 1995 (Khachik, U.S. Pat. No. 5,382,714). The saponifiedMarigold oleoresin was obtained from Kemin Industries (Des Moines, Iowa)and is normally prepared from extraction of dried Marigold petals withn-hexane, followed by saponification and solvent evaporation. To date,this process is the only available method for isolation and purificationof lutein (containing 3-6% zeaxanthin) from Marigolds in puritiesgreater than 97%. Recently, another process for the isolation of luteinfrom a saponified Marigold oleoresin has been reported wherein lutiencan be obtained in 70-85% purity (U.S. Pat. No. 5,648,564, 1997). Thisprocess employs propylene glycol (40.9% weight percent) and an aqueousalkali (18.2% weight percent) to saponify a hexane extract of driedMarigold petals (marigold oleoresin, 40.9% weight percent) containinglutein esters at 70° C. in 10 hours.

There are several major disadvantages with this process; these arediscussed as follows. The Marigold oleoresin is prepared by extractionof dried marigold petals in boiling n-hexane for extended periods oftime. Since lutein/zeaxanthin, and carotenoids in general, are sensitiveto heat treatment, this procedure can result in degradation orisomerization of these compounds. Furthermore, according to the Food andDrug administration's (FDA) Federal Register Documents, Code of FederalRegulations & Food, Drug, and Cosmetic act, n-hexane is considered amongthe solvents whose levels in foods and pharmaceutical products should belimited. The classification of organic solvents by the FDA will bedescribed later in this text.

In the next step of this process, the hydrolysis of lutein andzeaxanthin esters in the marigold oleoresin is conducted in an aqueoussolution in the presence of alcohol and propylene glycol in which thefatty acid esters of lutein and zeaxanthin have very low solubility. Asa result, this process requires high temperatures of up to 70° C. and 10hours to complete the saponification. This can once again result in thedegradation and isomerization of lutein and zeaxanthin.

Due to the high viscosity of propylene glycol, during handling andseveral purification steps, the saponified product is additionallysubjected to high temperatures ranging from 70 to 85° C. Thisun-necessary exposure to heat in the presence of air can result inoxidative degradation of lutein and zeaxanthin and formation of a numberof side products.

In summary, the above patented process (U.S. Pat. No. 5,648,564) employsextraction and saponification of Marigolds in two separate steps whichare then followed by several purification steps. According to theauthors, when the extraction and saponification steps were combined tosimplify the procedure, the result was a 64.7% reduction in the yield oflutein in comparison to the two-step extraction and saponificationprocesses described above. Overall, these procedures are quitetime-consuming, are carried out under harsh conditions, and producelutein in only 70-85% purity.

The earlier patent by the inventor (Khachik, U.S. Pat. No. 5,382,714)generally has two major disadvantages. This process also uses n-hexaneas the extracting solvent and, at the last purification step, it employsdichloromethane and n-hexane as the recrystallization solvents to obtainlutein containing 3-6% zeaxanthin in purities of 97% or greater. Sinceaccording to the FDA, the use of dichloromethane and hexane in drug andfood products should be limited, the lutein purified by these solvents,should be thoroughly dried under high vacuum to remove residualsolvents.

The process described here provides a convenient and economical route tolutein, zeaxanthin, and several minor carotenoids by employing asimultaneous extraction and saponification procedure at room temperaturefor only a few hours. Most importantly, this process addresses all thedisadvantages and concerns with regard to all of the previously patentedprocedures described above. As a result, the lutein (from Marigolds) andzeaxanthin (from LCM berries) obtained by this process are in purity of97% or greater and are therefore suitable for human consumption. Thisextraction and purification process has also been successfully employedfor isolation of lutein and several minor carotenoids from green plantswhich serve as an alternative source for commercial production oflutein.

BRIEF SUMMARY OF THE INVENTION

This invention employs a simultaneous extraction and hydrolysis(saponification) procedure for isolation and purification of lutein,zeaxanthin, and several rare carotenoids from Marigold flowers, LCMberries, and green plants. The plants are mixed with tetrahydrofuran(THF) and sufficient amounts of ethanol or methanol (preferably foodgrade ethanol) with the addition of either 5% or 10% potassium or sodiumhydroxide to maintain the pH at 12 and the mixture is homogenized atroom temperature for 1 to 2 hours. This process not only extractscarotenoids from plants under mild conditions, within a few hours, butit simultaneously hydrolyses the fatty acid esters of majordihydroxycarotenoids such as lutein and zeaxanthin as well asmonohydroxycarotenoids such as alpha-cryptoxanthin andbeta-cryptoxanthin which are present in minute quantities. As a result,the free and un-esterified forms of these hydroxycarotenoids areregenerated.

In the case of green plants, in which lutein is present in its freeform, the simultaneous extraction and saponification results in theconversion of chlorophylls to their water soluble derivatives and allowsthe removal of these compounds from lutein, beta-carotene, and othercarotenoids. In the next step, the mixture is filtered off, the solvents(THF and alcohol) are evaporated, and the residue is washed with wateruntil the pH of the aqueous wash is neutral (pH=7.0). This treatmentremoves the salts of fatty acid esters, the base (potassium or sodiumhydroxide), and the water soluble plant components. Aftercentrifugation, depending on the plant source, the resulting crystals oflutein or zeaxanthin are approximately 70% pure and contain minorquantities of other carotenoids. In the final purification step, luteinor zeaxanthin are dissolved in minimum amount of THF, and water is addeduntil the solution becomes cloudy; at this point the recrystalliztion ofthese compounds takes place. The mixture is then centrifuged and thesolid crystals are washed with water or alcohol to remove the THF. Afterdrying under high vacuum, the crystalline lutein or zeaxanthin areobtained in purities of 97% or greater.

Alternatively, 70% lutein or zeaxanthin may be purified by passing asolution of these compounds in THF and water through a column packedwith n-silica gel. After collection of the fraction containing lutein orzeaxanthin (greater than 97% purity), the THF is evaporated and theresidue is washed with water or alcohol and dried as described above. Asseparate fractions, several rare carotenoids such as alpha-cryptoxanthinand beta-cryptoxanthin (Marigolds and LCM berries) as well as a mixtureof lutein, beta-carotene, neoxanthin, violaxanthin, and lutein epoxide(from greens) are also obtained in high purity.

The processes described above is a convenient and commercially viablemethod for extraction and isolation of lutein and zeaxanthin from plantsources in which these compounds are esterified with fatty acids.Similarly this method can be applied to green plants where the presenceof chlorophylls complicate the isolation of the un-esterified lutein. Inaddition to purifying lutein and zeaxanthin to 97% or greater, thisprocess also allows the isolation of several rare carotenoids which arenot commercially available. The production of carotenoids according tothis process can be conducted under controlled and mild conditions atroom temperature or below 40° C. to avoid the isomerization anddegradation of these heat-sensitive compounds. The only organic solventused is THF which, due to its solubility, can be easily removed bywashing with water and/or alcohol. Consequently, the carotenoidsproduced according to these procedures can be safely used as nutritionalsupplements or food coloring additives.

DETAILED DESCRIPTION OF THE INVENTION Nomenclature

For convenience, the trivial rather than the correct systematic names ofcarotenoids have been used throughout this text. The chemical structuresof lutein [(3R,3′R,6′R)-lutein], zeaxanthin [(3R,3′R)-zeaxanthin],alpha-cryptoxanthin [(3R,6′R)-alpha-cryptoxanthin], andbeta-cryptoxanthin [3R-beta-cryptoxanthin], isolated from Marigoldflowers (Tagete erecta) and berries of Lycium Chinese Mill, have beenestablished to be identical with the dietary forms of these compoundsfound in most fruits and vegetables. The terms all-E and Z isomers ofcarotenoids refer to all-trans and cis isomers of these compounds. Forin-chain geometrical isomers of carotenoids, the terms all-trans andcis, which have been used with the old nomenclature, are no longerappropriate. If not specified, the terms lutein or zeaxanthin refer tothe most common geometrical forms (all-E or all-trans) of thesecarotenoids in plants. The use of the term “lutein esters” or“zeaxanthin esters” refers to either mono- or di- esters withoutlimitation.

The present invention applies a simultaneous extraction andsaponification process to three generally different plants sources forisolation of lutein and zeaxanthin; these are: dried petals of Marigolds(Tagete erecta), LCM berries (Lycium Chinese Mill), and several greenvegetables such as Kale (Brassica oleracia, var. acephala), Spinach(Spinacia oleracia), and Collard green (Brassica oleracia, var.Champion). These plant sources of carotenoids are described as follows.

The dried petals of marigold flowers, are harvested and prepared inCentral America and are imported into the U.S. Flowers are hand-pickedand ensiled to preserve them until they can be economically dried. Themarigold blossoms are then placed in a freeze drying apparatus and aredehydrated under controlled conditions. After dehydration, the flowersare put through a series of air separators and mechanical separatorswhere the petals are separated from any other materials and converted toa homogenous Marigold meal. The carotenoids in Marigold meal areesterified with fatty acids such as lauric, myristic, and palmitic acid,however, upon hydrolysis the parent hydroxycarotenoids are regenerated.As shown in a previous patent by the author (Khachik, 1995, U.S. Pat.No. 5,382,714, herein incorporated by reference), the majorhydroxycarotenoid in Marigold is lutein which is normally accompanied by3-6% of its isomeric compound, zeaxanthin.

LCM berries are normally grown in China and can be obtained from most ofthe Chinese supermarkets across the U.S. However, a variety of thisfruit is also currently grown on a commercial scale by Rehnborg Centerfor Nutrition in Lakeview, Calif. For the first time in 1995, the authorisolated several grams of zeaxanthin from LCM berries for a humansupplementation study and demonstrated that this plant is an excellentsource of zeaxanthin (Khachik et al., J. Cellular Biochem. 1995,22:236-246). However, the details of the isolation and purification ofzeaxanthin was not published. Zeaxanthin in LCM berries is mostlyesterified with palmitic acid and only trace amounts of this compound isesterified with lauric and myristic acids. LCM berries do not containany measurable amount of lutein but several other carotenoids such asalpha-cryptoxanthin, beta-cryptoxanthin, and beta-carotene are presentin this fruit in minute quantities. For the present study, largequantities of the berries were purchased from a local Chinesesupermarket.

In a 1986 publication by the author it has been shown that greens arealso a good source of lutein and beta-carotene as well as several rarecarotenoids such as neoxanthin, violaxanthin, and lutein epoxide(Ehachik et al. J. Agric. Food Chem., 1986, 34: 603-616). Furthermore,the analysis of several green vegetables indicated that the higher thelevels of chlorophylls, the higher were the levels of carotenoids.Although in greens, lutein is not esterified and is present in its freeform, simultaneous extraction and saponification by the present processconverts the chlorophylls to their water-soluble derivatives and allowsthe isolation and purification of carotenoids. No measurable amount ofzeaxanthin can normally be found in green plants. For the presentinvention, Kale, Spinach, and Collard green were purchased from a localsupermarket. These vegetables were selected due to their relatively highlutein content in compare to other common green vegetables. As shown bythe author previously, in green plants the general profile ofcarotenoids are same and the only variations are in concentrations ofindividual carotenoids. Therefore, the present process can be similarlyapplied to other dark green plants which may also serve as aneconomically viable source for isolation of lutein.

In the present invention, the choice of tetrahydrofuran (THF) as theextracting solvent was based on a guideline set by the Department ofHealth and Human Services, Food and Drug Administration (FDA) in DocketNo. 97D-0148 published in Federal Register: May 2, 1997 (volume 62,Number 85, pages 24301-24309). The draft guideline entitled “Impurities:Residual Solvents” and was prepared under the auspices of theInternational Conference on Harmonization (ICH) of TechnicalRequirements for Registration of Pharmaceuticals for Human Use. Thedraft guideline recommends acceptable amounts of residual solvents inpharmaceuticals for the safety of the patient as well as recommendingthe use of less toxic solvents in the manufacture of drug substances anddosage forms. According to this guidelines, solvents are divided intothree classes. These are:

Class 1: Solvents to be avoided. Known human carcinogens, stronglysuspected human carcinogens, and environmental hazards.

Class 2: Solvents to be limited. Nongenotoxic animal carcinogens orpossible causative agents of other irreversible toxicity such asneurotoxicity or teratogenicity; solvents suspected of other significantbut reversible toxicities.

Class 3: Solvents with low toxic potential to man. No health basedexposure limit is needed. Class 3 solvents have Permitted Daily Exposure(PDE) of 50 milligrams (mg) or more per day.

Ethanol and THF employed by the present invention are listed by the FDAin Class 3 and are therefore quite safe for commercial production ofcarotenoids for human use. The advantage of THF in compare to otherorganic solvents of Class 3 is due to the strong solubility ofcarotenoids in this solvent which allows the extraction of thesepigments from the matrices of various plants in an efficient and speedymanner. In addition, THF is water soluble and therefore can be used in ahomogenous phase extraction of carotenoids from plants in whichsignificant amount of water may be present (Kale, Spinach, Collardgreen). The solubility strength of THF for carotenoids and thehomogeneity of THF, ethanol, and water allow for the simultaneousextraction and saponification of carotenoids from plants. This isbecause the extracted carotenoids in a homogenous phase can be readilyhydrolyzed at room temperature within several hours whereas in aheterogenous phase immiscible solvents such as a mixture of hexane, analcohol, and water, would require high temperatures over extended periodto accomplish the hydrolysis. The heterogeneous phase hydrolysis ofcarotenoids can present a serious problem especially in scaling up forcommercial production. In such cases, the unavoidable high temperaturesused for hydrolysis can increase the risk of oxidative degradation andisomerization of carotenoids.

In a typical process the Marigold meal (100 g), tetrahydrofuran (THF,1000 ml), and sodium- or potassium hydroxide (25 g) in food gradeethanol (250 ml) are homogenized at room temperature. The homogenate issteeped at an elevated pH of about 11-14, preferably 12, for about twohours. Although food grade ethanol is preferred, other alcohols such as1-butanol, 2-butanol, 2-methyl-1-propanol, 1-propanol, and 2-propanolwhich are all listed as safe in Class 3 solvents by the FDA may also beused. Preferred solvents are also selected for their boiling points.Solvents with boiling points of about 75° C. to 120° C. are preferred.Ethanol, with a boiling point of 78° C., is most preferred. The pH ofthe mixture is monitored and automatically maintained at about pH 12. Anadditional amount of sodium- or potassium hydroxide (25 g) in 250 ml ofethanol is normally needed to maintain the pH at 12. The extraction andsaponification is completed in about 2 hours. The solvents are thenevaporated and the residue is washed with 1000 ml of a 1/1 mixture waterand alcohol to remove the base and the water soluble components. Thelutein crystals are collected by centrifugation, washed with alcohol(100 ml), and dried under high vacuum overnight at room temperature. Thelutein crystals (2.0 g) obtained are about 70% pure and can berecrystallized from a mixture of THF (20 ml) and water (30 ml) and driedunder high vacuum overnight to give 1.2 g of lutein containing about 5%zeaxanthin in 97% purity.

Alternatively, the above 70% pure lutein can also be purified by passinga solution of this compound in THF/water (1.5/1.0) through a column ofn-silica gel and subsequent of washing of the column with this solventmixture. After collection of the lutein fraction, the THF is evaporated,the crystals of pure lutein are collected by centrifugation, and washedwith 20 ml of an alcohol, preferably ethanol, to remove the residualwater from the product. Drying under high vacuum gives 1.2 g of 97% purelutein containing about 5% zeaxanthin. In another fraction from thispurification, about 0.3 g of a mixture of lutein, anhydrolutein,alpha-cryptoxanthin, and beta-cryptoxanthin is also obtained.

When the simultaneous extraction and saponification process forisolation of lutein from Marigold was compared to a two-steps process,in which the plant was first extracted and then saponified; the resultswere identical.

The simultaneous extraction and saponification was also successfullyapplied to the isolation of 97% pure zeaxanthin (0.48 g) from LCMberries (455.5 g) and a mixture of lutein, beta-carotene, neoxanthin,violaxanthin, lutein epoxide (0.7 g) from Kale (1800 g).

SIMULTANEOUS EXTRACTION AND SAPONIFICATION EXAMPLE 1 Isolation ofCarotenoids from Marigolds

Marigold meal (100 g) was vigorously homogenized in a blender withtetrahydrofuran (THF, 1000 ml) and 10% ethanolic KOH (250 ml) at roomtemperature. The pH of the solution was kept at 12 by monitoring theextraction and hydrolysis with an automatic pH-meter. During the courseof the extraction an additional 250 ml of 10% ethanolic KOH was neededto maintain the pH at 12. It is essential to avoid the addition ofexcessive amounts of the base to the mixture during the extraction andhydrolysis. This is because at a later purification step, the removal ofthe excess base will then require additional and un-necessary washing ofthe residue with large volumes of water which can be time consuming. Thecourse of the extraction and hydrolysis was monitored by Thin LayerChromatography (TLC) using hexane/acetone=90/10 on n-silica gel platesas well as by High Performance Liquid Chromatography (HPLC) according toa published procedure by the author (Khachik et al. J. Chrom. 449:119-133, 1988). After 2 hours, carotenoid esters were shown to becompletely hydrolyzed. The mixture was filtered off and the solids werewashed with THF (1000 ml). The solvents were evaporated under reducedpressure at approximately 40° C. to almost dryness and the solids werestirred at room temperature with a 1/1 mixture of water and food gradeethanol (700 ml) for 10 minutes. The mixture was then centrifuged andthe water/alcohol wash was removed. The washing of the solids wasrepeated 2 more times at which point all the base was shown to have beenremoved and the pH of the aqueous wash was at pH=7. The solids werewashed with 100 ml of ethanol, centrifuged, and dried under high vacuumat room temperature overnight to give lutein as yellow crystals (2.0 g,70% pure lutein). The HPLC analysis (Khachik et al. J. Chrom. Biomed.Appl. 582: 153-166, 1992) of the lutein crystals at this point revealedthe carotenoid composition shown in Table I.

TABLE I Carotenoid Composition of 70% Pure Lutein Isolated from anExtract of Marigold Flowers Carotenoids Composition (%) all-E-Lutein93.0 Total-Z-Luteins 0.3 all-E-Zeaxanthin 5.4 Anhydroluteins 0.8alpha-Cryptoxanthin 0.2 beta-Cryptoxanthin 0.3

Purification of Lutein by Recrystallization

The lutein crystals (2.0 g, 70% pure) were dissolved in THF (20 ml) andwater (30 ml) was added to commence recrystallization. The mixture wasstirred at room temperature for 10 minutes and the solids were separatedby centrifugation. After removal of the solution, the lutein crystalswere washed with ethanol (20 ml), centrifuged, and dried under highvacuum overnight at room temperature. This gave 1.2 g of lutein whichwas shown by HPLC-UV/Visible photodiode array detection coupled withMass Spectrometry (MS) to be 97% pure but contained about 4% ofall-E-zeaxanthin (Khachik et al. J. Chrom. Biomed. Appl. 582: 153-166,1992).

The THF/water solution from the above recrystallization was evaporatedto remove the THF and to precipitate small amounts of lutein along withother carotenoids (0.4 g) which were separated by centrifugation. Themixture of carotenoids were washed with small amounts of ethanol (about4 ml) and dried under high vacuum. HPLC analysis of the dried solidshowed that in addition to all-E-lutein, this fraction was enriched in13,13′-di-Z-lutein (stereoisomer of lutein) as well as anhydrolutein,alpha-cryptoxanthin, and beta-cryptoxanthin.

Purification of Lutein and Other Carotenoids on a n-Silica Gel Column

The 70% pure lutein (2.0 g) was dissolved in THF (20 ml) and was passedthrough a glass column of n-Silica gel (15 cm L X 4 cm I.D.) packedunder slight air pressure using THF/water (1.5/1.0). The column waswashed with 200 ml of THF/water (1.5/1.0) and fractions 1 (50 ml) and 2(150 ml) were collected. After evaporation of THF under reduced pressureat 40° C, and removal of water by centrifugation, fractions 1 and 2 werewashed with 4 ml and 20 ml of ethanol, respectively, and dried underhigh vacuum at room temperature overnight. The first fraction (0.3 g)was shown by HPLC-UV/Vis-MS to be a mixture of anhydrolutein,alpha-cryptoxanthin, and beta-cryptoxanthin and the second fraction (1.2g) was shown to be pure all-E-lutein (97%) but contained approximately4% zeaxanthin.

EXAMPLE 2 Isolation of Carotenoids from Lycirm Chinese Mill (LCMBERRIES)

LCM berries (455.5 g) were rehydrated in a beaker containing 1000 ml ofwater overnight at room temperature. The water was decanted off and theLCM berries were vigorously homogenized with THF (2000 ml) and 10%ethanolic KOH (250 ml) at room temperature. The pH of the solution wasmaintained at 12 by monitoring the extraction and hydrolysis with anautomatic pH-meter. During the course of the extraction an additionalamount of 250 ml of 10% ethanolic KOH was needed to keep the pH at 12.The course of the extraction and hydrolysis was monitored by Thin LayerChromatography (TLC) using hexane/acetone=90/10 on n-silica gel platesas well as by High Performance Liquid Chromatography (HPLC) according toa published procedure by the author (Khachik et al. J. Chrom. 449:119-133, 1988). After 2 hours, carotenoid esters were shown to becompletely hydrolyzed. The mixture was filtered off and the solids werewashed with THF (with about 500 ml). The solvents were evaporated underreduced pressure at approximately 40° C. to almost dryness and thesolids were stirred at room temperature with a 1/1 mixture of water andfood grade ethanol (about 100 ml) for about 10 minutes. The mixture wasthen centrifuged and the water/alcohol wash was removed. The washing ofthe solids was repeated 2 more times at which point all the base wasshown to have been removed and the pH of the aqueous wash was at pH=7.The solids were washed with 30 ml of ethanol, centrifuged, and driedunder high vacuum at room temperature overnight to give zeaxanthin asorange crystals (0.48 g, 75% pure zeaxanthin). The HPLC analysis(Khachik et al. J. Chrom. Biomed. Appl. 582: 153-166, 1992) of thezeaxanthin crystals at this point revealed the carotenoid compositionshown in Table II.

TABLE II Carotenoid Composition of 75% Pure Zeaxanthin Isolated from anExtract of Lycium Chinese Mill Carotenoids Composition (%)all-E-Zeaxanthin 91.0 Total-Z-Zeaxanthins 5.8 alpha-Cryptoxanthin 1.1beta-Cryptoxanthin 1.3 beta-Carotene 0.8

Purification of Zeaxanthin by Recrystallization

The zeaxanthin crystals (0.48 g, 75% pure) were dissolved in THF (5 ml)and water (about 10 ml) was added to commence recrystallization. Themixture was stirred at room temperature for about 10 minutes and thesolids were separated by centrifugation. After removal of the solution,the zeaxanthin crystals were washed with ethanol (about 5 ml),centrifuged, and dried under high vacuum for about 8 hours at roomtemperature. This gave 0.35 g of zeaxanthin which was shown byHPLC-UV/Visible photodiode array detection coupled with MassSpectrometry (MS) to be 97% pure but contained about 3.5% ofZ-zeaxanthins (Khachik et al. J. Chrom. Biomed. Appl. 582: 153-166,1992).

The THF/water solution from the above recrystallization was evaporatedto remove the THF and to precipitate small amount of zeaxanthin alongwith other carotenoids (0.1 g) which were separated by centrifugation.The mixture of carotenoids were washed with small amount of ethanol (1ml) and dried under high vacuum. HPLC analysis of the dried solid showedthat in addition to zeaxanthin, this fraction was enriched inalpha-cryptoxanthin, beta-cryptoxanthin, and beta-carotene.

EXAMPLE 3 Isolation of Carotenoids from Greens

The general procedure described here for the isolation of carotenoidsfrom Kale was similarly applied to spinach and collard green.

Fresh kale (1800 g) was placed in a Robot-que and cut into small peace.This was vigorously homogenized with THF (5000 ml) and 10% ethanolic KOH(500 ml) at room temperature. The pH of the solution was maintained at12 by monitoring the extraction and hydrolysis with a pH-meter. Duringthe course of the extraction an additional amount of 500 ml of 10%ethanolic KOH was needed to maintain the pH at 12. The course of theextraction and hydrolysis of chlorophylls to their derivatives wasmonitored by Thin Layer Chromatography (TLC) as well as by HighPerformance Liquid Chromatography (HPLC) according to a publishedprocedure by the author (Khachik et al. J. Agric. Food Chem. 34:603-616,1986). After 2 hours, chlorophylls were completely hydrolyzed to theirwater soluble derivatives. The mixture was filtered off and the solidswere washed with THF (1000 ml). The solvents were evaporated underreduced pressure at approximately 40° C. to almost dryness and thesolids were stirred at room temperature with a 1/1 mixture of water andfood grade ethanol (100 ml) for 10 minutes. The mixture was thencentrifuged and the water/alcohol wash was removed. The washing of thesolids was repeated 4 more times at which point all the base and thegreen chlorophyll derivatives were removed and the pH of the aqueouswash was at pH=7. The solids were washed with 30 ml of ethanol,centrifuged, and dried under high vacuum at room temperature overnightto give a mixture of carotenoids (0.923 g). This was dissolved in 5 mlof THF and treated with 10 ml of water to recrystallize carotenoidswhich were separated by centrifugation. After removal of the solvents,the crystals were washed with ethanol (5 ml) and dried under high vacuumovernight at room temperature. The HPLC analysis (Khachik et al. J.Agric. Food Chem. 34: 603-616, 1986) of the mixture (0.7 g) revealed thecarotenoid composition shown in Table III.

TABLE III Carotenoid Composition of a Purified Extract from KaleCarotenoids Composition (%) all-E-Lutein 48.7 Total-Z-Luteins 2.3beta-Carotene 19.0 Neoxanthin 16.0 Violaxanthin 11.0 Lutein Epoxide 3.0

From the above examples it can be seen that the present invention hasaccomplished the isolation of lutein and zeaxanthin as well as a mixtureof several rare carotenoids from plants in substantially pure form undercontrolled and mild condition to preserve the integrity of theseessential dietary compounds. This economically viable process employssolvents which are not toxic and as a result, the carotenoids purifiedby this procedure can be safely used as nutritional supplements or foodcoloring additives.

What is claimed is:
 1. A method for the extraction and saponification oflutein esters or zeaxanthin esters from a plant source containing luteinesters or zeaxanthin esters, comprising; extracting lutein esters orzeaxanthin esters from said plant source by contacting the plant sourcewith a solution containing tetrahydrofuran and an alcohol to obtain amixture; saponifying the mixture; removing the tetrahydrofuran andalcohol from the mixture to obtain a residue; washing the residue withan aqueous solution; and isolating lutein or zeaxanthin as crystals. 2.The method of claim 1 wherein extraction and saponification areconducted simultaneously.
 3. The method of claim 1 wherein the alcoholis selected form the group consisting of ethanol, 1-butanol, 2-butanol,2-methyl-1-propanol, 1-propanol, and 2-propanol.
 4. The method of claim3 wherein the alcohol is ethanol.
 5. A method for the simultaneousextraction and hydrolysis of lutein esters from marigold petals,comprising: extracting and saponifying lutein esters from said petals bycontacting said petals with a solution comprising tetrahydrofuran and analcohol at an elevated pH to obtain a mixture comprising hydrolyzedlutein; removing unwanted solids from the mixture to obtain a liquidsportion; removing the tetrahydrofuran and alcohol from the liquidsportion to obtain a residue comprising lutein free from esters; washingthe residue with an aqueous mixture; and collecting lutein crystals. 6.The method of claim 5 wherein the alcohol is selected form the groupconsisting of ethanol, 1-butanol, 2-butanol, 2-methyl-1-propanol,1-propanol, and 2-propanol.
 7. The method of claim 5 further comprisingwashing the collected lutein crystals with an alcohol having a boilingpoint between 75° C. and 120° C., and drying the crystals.
 8. The methodof claim 7 further comprising recrystalizing the lutein crystals from anaqueous mixture of tetrahydrofuran.
 9. The method of claim 5 wherein thepH is monitored during the simultaneous extracting and saponifying stepsand the pH is adjusted by adding base.
 10. The method of claim 5 whereinsaponification (hydrolysis) occurs during the extraction step and thebase is NaOH or KOH.
 11. The method of claim 5 wherein the marigoldpetals, prior to extraction, are milled to a meal.
 12. The method ofclaim 5 wherein the pH is about
 12. 13. The method of claim 1 whereinthe extracting and saponifying steps are conducted at about roomtemperature in two hours.
 14. A method for the simultaneous extractionof lutein and conversion of chlorophylls to water soluable derivitivespresent in green leafy vegetables, comprising: extracting lutein andhydrolyizing chlorophylls from said vegetables by contacting saidvegetables with a solution comprising tetrahydrofuran and an alcohol atan elevated pH to obtain a mixture; removing solids from the mixture toobtain a liquid extract; removing the tetrahydrofuran and alcohol fromthe liquid extract to obtain a residue; washing the residue with anaqueous mixture to remove the base and water soluble chlorophyllderivatives; and collecting lutein.
 15. The method of claim 2 whereinthe plant source is Lycium Chinese berries or marigold petals or greenleafy vegetables.
 16. A method for the simultaneous extraction andsaponification of one or more carotenoids from a plant sourcecomprising: steeping the plant source in the presence of only class 3solvents, at room temperature, to obtain an extract comprisingcarotenoid extraction products.
 17. The method of claim 16 whereinsolvents for extraction and saponification are tetrahydrofuran, and analcohol selected from the group consisting of ethanol, 1-butanol,2-butanol, 2-methyl-1-propanol, 1-propanol, and 2-proponol.
 18. Themethod of claim 16 wherein the solvent is a mixture of tetrahydrofuranand ethanol, and extraction/saponification is conducted in the presenceof KOH.
 19. The method of claim 16 wherein the extraction/saponificationis conducted at a pH of about
 12. 20. The method of claim 18 wherein theextraction/saponification is conducted at room temperature for twohours.
 21. The method of claim 1 further comprising purifying crystalsby dissolving the crystals to create a solution and passing the solutionof lutein or zeaxanthin through a column packed with n-silica gel andisolating pure lutein or zeaxanthin.
 22. A method for isolating luteincomprising simultaneously extracting and saponifying lutein esters.